To provoke the specific expression of certain genes in the anthers of transgenic plants, different promoters have been isolated, characterised and used. The expression of these promoters is specific to a given tissue of the anther. Some of them have been used in cell ablation techniques to produce sterile plants of great utility for the production of hybrid seeds. Currently, genetic ablation of plant cells is being used to carry out studies of cell functioning and signalling within specific organs or tissues and to generate androsterility (male-sterility).
Genetic ablation is based on the induction of cell death by means of the expression of any enzyme that is able to destroy cell integrity such as proteases, lipases and RNases (for example, barnase and T1 RNase). Equivalent results can be obtained by expressing toxic substances into the cells [Day C D and Irish V F. Trends Plant Sci., 2: 106–111, 1997]. An example of this last method is the use of peptides that deactivate ribosomes, as is the case of the A chain of the toxin from Corynebacterium diphtheria (DTA) and the exotoxin A from Pseudomonas aeruginosa. Several groups have suggested the production of androsterility via overproduction of growth regulators. The synthesis of auxins and cytokinines using genes of non-plant origin, such as genes 1 and 2 of the Ti plasmid, or the genes rol A, B and C of the plasmid Ri, represent methods where these factors become toxins due to the magnitude and inappropriate moment of expression.
Methods have also been developed that do not directly destroy the tissue, but rather give rise to cells susceptible to specific ablative agents. An example of this approach is the use of an “antisense” RNA from a previously established gene. This confers inherited resistance to a chemical agent, such as, for example, tolerance to a herbicide (Fabijanski SF et al., In vitro Cell. Dev. Biol. 28: 46–52, 1992]. The effect of the “antisense” RNA is to specifically eliminate the chemical resistance, for example, in pollen, so that the application of the herbicide leads to destruction of the pollen. This method converts a herbicide into a gametocide.
In order to obtain an efficient genetic ablation, it is crucial to have a cytotoxic gene that acts only where it is expressed and to have an appropriate promoter that controls the spatial-temporal expression of the cytotoxic gene. For this reason, the characterisation of active gene promoters in different cell types and/or at different moments during differentiation of the anther has allowed to examine the functions of different tissues of the anther during the gametogenesis by means of cell ablation [Mariani C et al. Nature, 347: 737–741, 1990; Paul W et al., Plant Mol. Biol., 19: 611–622, 1992; Hird D L et al., Plant J., 4: 1023–1033, 1993], with particular emphasis on the cell to cell interactions [Roberts M R et al., Sex. Plant Reprod., 8: 299–307, 1995]. Similarly, this technique is being used by large seed producing companies for the development of androsterility, a desirable feature in the processes for obtaining hybrid seeds [Williams M E, Trends Biotechnol., 13: 344–349, 1995].
In several works, the function of cell to cell interactions has been analysed during the development of the reproductive structures. For example, several different promoters have been used to direct the expression of a cytotoxic gene in cells from tapetum in the anther with the object of determining the effect of ablation on the development of pollen [Mariani C et al., Nature 347: 737–741, 1990; Roberts M R et al., Sex. Plant Reprod. 8:299–307, 1995]. Ablation of tapetal cells in different studies has different effects on the development of pollen. The use of a specific promoter of tobacco tapetal cells (TA29) directing the expression of the barnase gene (ribonuclease) during the tetrad phase of the pollen development leads to androsterility, which indicates that the tapetum is essential for the production of viable pollen at this stage (Mariani C et al., Nature 347: 737–741, 1990). On the contrary, the substitution of the TA29 promoter by the APG promoter from Arabidopsis, specific to tapetur in the microspore phase of pollen, does not have any effect on the pollen [Roberts M R et al., Sex. Plant Reprod. 8:299–307, 1995]. This latter datum indicates that the tapetum is not essential for the formation of pollen from the disintegration of the microspore tetrads. A histochemical analysis of the anther development in transgenic plants of Brassica with the TA29-barnase construct showed the degradation of RNA within the tapetal cells along with the disappearance of RNA from the microspores [De Block M and Debrouwer D, Planta 189: 218–225, 1993]. This observation suggests that the microspores remain permeable to small molecules after initiation of the deposition of sporopollenine and in late phases of their development, since the TA29 promoter does not direct the expression of genes in microspores.
Beals T P and Goldberg R B [Plant Cell, 9: 1527–1545, 1997] put into practice a new cell ablation strategy for determining what cell types from an anther are implicated in the dehiscence process. They transformed tobacco plants with two constructs: the construct formed by the TA56 promoter, active in the septum, in the stomium and in the connective tissue, and the barnase gene along with one of the following constructs in an alternative form: a) the TP12 promoter, active in most of the tissues of the anther, along with the barstar gene (barnase inhibitor), b) the TA20 promoter, active in most of the tissues of the anther but with a different distribution pattern from that of TA12, and the barstar gene and c) the soybean lectin gene promoter, active in the cells of the connective tissue, the septum and the stomium, in addition to the barstar gene. The analysis of the different phenotypes of the transgenic plants showed that the dehiscence process only depends on the presence of a functional stomium.
Shull [J. Ind. Abst. Vererb. 12: 97–149, 1914] was the first one to introduce the term heterosis to describe the advantages offered by heterozygosis regarding cell division, growth and other physiological activities of an organism. The result of these advantages: increase in size, vigour, yield, an earlier fructification and resistance to diseases has for some decades induced the attempts to obtain hybrid varieties [Tsaftaris S A, Physiol. Plantarum 94: 362–370, 1995]. Most plants containing both male and female reproductive organs self-pollinate themselves (corn, rice, soybean, tomato, etc.), which causes problems in the processes for producing hybrid seeds [Kriete G et al., Plant J. 9: 809–818, 1996]. To avoid this problem, a system has to be used that controls the self-pollination. This system may be mechanical, chemical or genetic.
The mechanical system consists of manually eliminating the anthers from flowers (emasculation), which is an arduous and expensive task.
The chemical method is based on the use of chemical products (gametocides) that specifically destroy the pollen leading to transitory androsterility. This approximation is not particularly effective in cultures with a long blooming period or with variable or uncontrollable blooming conditions. In addition, the commercial production of hybrid cells via gametocides is characterised by being expensive and by the high relative effectiveness of the chemical products.
Most commercial systems for the production of hybrid seeds are based on genetic methods to control the blooming, so that mutually incompatible plants or androsterile plants are used, that is, plants that do not produce pollen, are unable to release the pollen or develop pollen that is unable to product self fertilisation [Homer H T and Palmer R G, Crop Sci. 35: 1527–1535, 1995]. The production of androsterile plants is of great utility for obtaining hybrid seeds. The male sterility eliminates the possibility of self-fecundation of the plant, thus facilitating the production of hybrids that find important applications in the genetic improvement programmes.
The methods for obtaining hybrid seeds described up until now have limitations and are not applicable in important cultures of agricultural interest. Currently, new strategies are being developed based on genetic engineering to produce androsterile plants [Gates P, Biotechnol. Genet. Engineering Rev. 13: 181–195, 1994]. The development of methods based on recombinant DNA and the characterisation of genes implicated in the development of pollen has allowed the proliferation of molecular systems that provoke nuclear male sterility (NMS), [Scott R J et al., Plant Sci. 80: 167–191, 1991]. As has already been mentioned, the androsterility in molecular systems is achieved by means of cell ablation processes preventing the development of the microspores or the tissues that lead to their development (tapetum and walls of the pollen sacs). In addition, it is possible to produce completely functional pollen, but this pollen is not released due to defects in the structure of the anther. To obtain androsterile plants useful in the production of hybrid seeds it is important to have a specific promoter for the tissues implicated in the development of the pollen, a system for selecting the androsterile line and a system for restoring the fertility in the F1 hybrid line.
Specific Promoters
The transcription process in most plant genes is controlled both temporally and spatially. The regulation of genetic activity is mediated by the interaction between trans factors and cis regulatory elements present in the promoter region of the gene. Thus, a promoter is a DNA sequence that directs the transcription of a structural gene and therefore is located in its 5′ region.
The genes that are exclusively expressed in the reproductive organs of the flower (stamen and carpels) are particularly interesting, as their promoters would potentially be able to direct the expression of other genes towards said organs and provoke male or female sterility in the flower. This is the case in some gene promoters that are specifically expressed in the anthers, which have already been used, in combination with ablative agents that only affect the developing pollen, in biotechnological approaches that aim to produce androsterile plants unable to self-pollinate. Therefore, these types of approach greatly depend on the existence of promoters providing a suitable expression. During the last years, numerous genes have been isolated and characterised that are specifically expressed in tissues and cells related to the development of pollen, therefore, promoters are available for this end [Scott R J et al., Plant Sci. 80: 167–191, 1991].
The promoters that are selected for expressing toxic agents or those that are degenerative for the tissue are usually promoters regulated by development, sufficiently active and specific for the target cells. If the target cell is tapetal tissue, the promoter should be active early in the development of the microspore in order to halt the process before the microspores become independent from the support of the tapetum. Similarly, the promoter should act before meiotic segregation to prevent the lack of degenerative activity in part of the microspores if the lethal gene is hemizygotic.
An alternative use of promoters regulated by development is the use of inducible promoters. However, the number of described promoters showing specific chemical induction is very small. The use of an inducible promoter for blocking the development of pollen has advantages when it comes to maintaining and increasing the female line because the plants are fertile and can be multiplied by self-crossing. Inherited active or inducible promoters can also be used if the androsterility is based on the suppression of a gene that is expressed in a tissue implicated in the development of pollen, for example, via “antisense”.
Selection of the Androsterile Line.
In order to produce hybrid seeds in industrial quantities it is necessary to increase the female line. Although it is possible to produce many androsterile plants by means of in vitro propagation, for the plants of agricultural interest, this would be too expensive. A common strategy for multiplying the androsterile line is to join a gene that confers resistance to a herbicide to the ablative gene [Mariani C et al., Nature 357: 384–387, 1992]. After crossing the androsterile line with an isogenic and fertile line, the plants that have not inherited the sterility are eliminated by the herbicide. By analogy, any gene allowing discrimination between the two phenotypes could be used, such as for example those affecting the pigmentation of the seeds.
Restoring Fertility
The restoration of fertility in hybrid plants can be carried out by crossing them with transgenic lines expressing an inhibitor specific to the toxic enzyme used for producing the androsterility, as is the case for the barstar gene, the product of which inhibits the action of barnase [Mariani C et al., Nature 357: 384–387, 1992] or an “antisense” RNA of the lethal gene used [Schmülling T et al., Mol. Gen. Genet. 237: 385–394, 1993].
Molecular Systems Used for Obtaining Androsterile Plants
The first ablation strategy designed for producing androsterility was proposed by Mariani et al. [Nature 347: 737–741, 1990]. The promoter of the TA29 tobacco gene, specific to tapetum, was used for directing the expression of two different RNases (T1 Rnase from Aspergillus oryzae and barnase from Bacillus amyloliquefaciens) in tobacco and Brassica napus. The obtained androsterile transgenic anthers lacked the tapetum and contained the pollen sacs with no microspores or pollen grains. The TA29-barnase construct was fused with the bar gene, a gene that confers tolerance to the ammonium gluphosinate herbicide, to allow selection of the androsterile plants in a population. The application of the herbicide to the progeny of a cross eliminates the fertile male plants and thus increases the efficiency with which the sterile plants can be isolated. Despite all this, these transgenic plants were no better than the spontaneous mutants if there was no possibility of reversing the androsterility. Mariani et al. [Nature 357: 384–387, 1992] solved this problem with the production of transgenic plants with a construct containing the inhibitor gene of ribonuclease barnase (barstar) under the control of the TA29 promoter. The TA29-barstar plants act as a restoring line and androfertile plants are obtained when they are crossed with plants transformed with the TA29-barnasa construct.
Apart from this system, the literature contains other methods for producing androsterile lines:
In Petunia hybrida, the nuclear androsterility was provoked by suppressing the synthesis of flavonoids in the anther, which prevents the maturation of the pollen. This was achieved in two different ways: through the “antisense” effect of RNA [van der Meer I M et al., Plant Cell 4: 253–262, 1992] and through a co-suppression [Taylor L P and Jorgensen R, J. Hered. 83: 11–17, 1992] of the chalconsynthetase gene, an enzyme implicated in the synthesis route of flavonoids. To restore fertility, flavonoids can be applied to the stigma or mixed with the pollen to allow the androsterile line to multiply by self pollination and therefore, homozygotic lines are obtained for the androsterile phenotype not requiring the use of any marker for selection [Ylstra B et al., Plant J. 6: 201–212, 1994].
In tobacco, the androsterility has also been induced by the expression of the gene rol C of Agrobacterium rhizogenes fused with the promoter CaMV 35S. Unfortunately, the androsterile phenotype was accompanied by other phenotypic alterations in the transgenic plant [Schmülling T et al., EMBO J. 7: 2621–2629, 1988]. The restoration of sterility was carried out by expression of an “antisense” RNA of the gene rol C in the F1 hybrids [Schmülling T et al., Mol. Gen. Genet. 237: 385–394, 1993].
O'Keefe et al. [Plant Physiol. 105: 473–482, 1994] described a system of inducible androsterility based on the expression of the P450SU1 cytochrome in tobacco tapetal cells. This protein is able to transform an inoffensive derivative of the R7402 gametocide, exogenously added, into its active form (500 times more toxic). However, possibly due to the fast metabolism of R7402, the androsterility is limited to flowers in a certain phase of development during the application of the compound. In addition, R7402 is itself toxic and begins to inhibit growth when it is applied in quantities four times greater than those used to produce androsterility in the classical way.
Another system for obtaining inducible androsterility is based on the use of the TA29 promoter of tapetum along with the argE gene from E. coli. The product of this gene deacetylises the N-acetyl-L-phosphinotrycin compound and transforms it into gluphosinate, a cytotoxic compound. When the N-acetyl-L-phosphinotrycin is applied to the tobacco plant, the tapetum degenerates and androsterile plants are obtained [Kriete G et al., Plant J. 9: 809–818, 1996]. Finally, we would like to point out that androsterility, in addition to being an important tool for obtaining hybrids, is also a desirable feature in plants capable to develop fruits in the absence of fertilisation (partenocarpic fruits). In this type of fruit, the seeds are absent and so the consumption or acceptance thereof by the consumers is increased [Rotino G et al., Nat. Biothecnol. 15: 1398–1401,1997]. Some partenocarpic cultures of agricultural interest are: pears, citric fruits, cucumber, grape and dates.